Alkynyl-substituted azasugar derivative and drug containing the same as the active ingredient

ABSTRACT

An alkynyl-substituted azasugar derivative and a drug containing the same as an active ingredient are disclosed. This drug is useful as a preventive or remedy for insulin-independent diabetes, rheumatoid arthritis, osteoarthritis, sepsis, acquired immune deficiency syndrome (AIDS), graft-versus-host disease (GVHD), asthma, atopic dermatitis, and ulcerative colitis.

TECHNICAL FIELD

The present invention relates to a hydroxamic acid derivative having anazasugar skeleton with acetylene-substituted arylsulfonylamide attached,which functions as a selective inhibitor against a TNF-α convertingenzyme (TACE).

BACKGROUND ART

TNF-α is a kind of cytokine produced by an active macrophage and servesas an inflammatory mediator at the inflammatory site. TNF-α isoriginally important cytokine. However, when it is excessively produced,it may cause diseases such as insulin-independent diabetes, rheumatoidarthritis, osteoarthritis, sepsis, acquired immune deficiency syndrome(AIDS), graft-versus-host disease (GVHD), asthma, atopic dermatitis, andulcerative colitis. Therefore, there is a fair chance that a drugcapable of inhibiting production of TNF-α (TNF-α production inhibitor)serves as a preventive or remedy for these diseases.

TNF-α is produced as a result of processing of a membrane-anchoredprecursor having a molecular weight of 26 kDa comprising 233 amino acidswith the converting enzyme (TACE). Therefore, intense interest has beenshown towards a compound having TACE inhibitory activity (TACEinhibitor) for the purpose of developing a TNF-α production inhibitor.

It has recently become apparent that a group of functional proteinsknown generically as ADAM (A Disintegrin And Metalloprotease) areinvolved in processing of various membrane-anchored proteins and also ithas been reported that ADAM 17 functions as TACE (see Non-PatentDocument 1). It is known that most MMP inhibitors, which have hithertobeen synthesized, also function as a TACE inhibitor because anextracellular matrix degrading enzyme, which has hitherto been known asa matrix metalloprotease (MMP), in a connective tissue is a relatedenzyme of ADAM molecules (see, for example, Patent Document 1).

However, it is pointed out that a MMP1 inhibitor causes arthralgia whenadministered to human (see Non-Patent Document 2). Therefore, a compoundcapable of selectively exerting an inhibitory effect on TACE is requiredas a TNF-α production inhibitor.

PCT International Publication (see Patent Document 2) discloses ahydroxamic acid derivative having an acetylene-substitutedarylsulfonylamide site as a TACE inhibitor. Various hydroxamic acidderivatives having a cyclic skeleton such as piperazine are disclosed.However, it is hardly to say that these derivatives have sufficientselectivity against TACE.

(Patent Document 1)

U.S. Pat. No. 5,691,382

(Patent Document 2)

PCT International Publication WO00/40729

(Non-Patent Document 1)

Black et al., Nature, 385, 729-733, (1997); Moss, Nature, 385, 733-736(1997)

(Non-Patent Document 2)

Scrip, No.2349, p. 20 (1998)

DISCLOSURE OF THE INVENTION

An object of the present invention is to find a compound capable ofexerting a selective inhibitory effect on TACE and to provide anexcellent TNF-α production inhibitor.

The present inventors have intensively studied and found that a compoundrepresented by the above formula (I) has a selective inhibitory effecton TACE, and thus the present invention has been completed.

The present invention will now be described in detail.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is directed to a compound represented by thegeneral formula (I):

wherein R¹ and R² represent a hydrogen atom, a C₁-C₈ alkyl group, aC₃-C₈ alkenyl group or a benzyl group and at least one of R¹ and R² is ahydrogen atom, and R³ represents a hydrogen atom or a hydroxyl group, ora pharmaceutically acceptable salt thereof, and a drug containing thesame as an active ingredient.

The C₁-C₈ alkyl group means a linear or branched alkyl group having 1 to8 carbon atoms and is particularly preferably a lower alkyl group having1 to 4 carbon atoms, such as methyl group or ethyl group.

The C₃-C₈ alkenyl group means a linear or branched alkenyl group having3 to 8 carbon atoms and is particularly preferably a lower alkenyl grouphaving 3 to 4 carbon atoms, such as allyl group or 2-methylallyl group.

Although plural asymmetric carbons exist in the compound (I), theasymmetric carbon constituting the piperidine ring is limited to thoserepresented by stereochemistry shown above. In case the asymmetriccarbon exists in an alkyl group selected as R¹ and R², all stereoisomersbased on the asymmetric carbon or a mixture thereof are included.

Compounds represented by the following formula (IIA) or (IIB):

wherein R¹′ and R²′ represent a hydrogen atom, a C₁-C₈ alkyl group, aC₃-C₈ alkenyl group or a benzyl group, R³ represents a hydrogen atom ora hydroxyl group, and CO—R⁴ represents a hydroxamic acid equivalent, arealso parts of the present invention.

As used herein, the hydroxamic acid equivalent means a monovalent atomicgroup —CONHOH in itself, and a functional group which can be chemicallyconverted into the atomic group —CONHOH by a conventional method. R⁴includes, for example, methoxy group, ethoxy group or benzyloxyaminogroup. Furthermore, since a benzyloxyamino group can be introducedeasily by the condensation reaction of carboxylic acid andbenzyloxyamine, the hydroxamic acid equivalent includes various estergroups which can be converted easily into a carboxyl group, in additionto the carboxyl group. Therefore, various carboxylic acids protectedwith a carboxyl protecting group are included, and specific examples ofR⁴ include benzyloxy group and tert-butyloxy group, in addition to theabove-mentioned methoxy group, ethoxy group and benzyloxyamino group.

Abbreviations and symbols used herein are as follows.

-   Ag₂O: silver oxide-   Bn: benzyl group-   CeCl₃: cerium chloride heptahydrate-   DMF: N,N-dimethylformamide-   DMP: 2,2-dimethoxypropane-   DMAP: 4-dimethylaminopyridine-   DIEA: diisopropylethylamine-   K₂CO₃: potassium carbonate-   THF: tetrahydrofuran-   Pd/C: palladium-carbon-   Pd(OH)₂/C: palladium hydroxide-carbon-   PPh₃: triphenylphosphine-   NaCN: sodium cyanide-   NH₂OH: aqueous 50% hydroxylamine solution-   TsOH: p-toluenesulfonic acid monohydrate-   MsCl: mesyl chloride-   TBAF: 1M tetrabutylammonium fluoride/THF solution-   TBDMS: tert-butyldimethylsilyl group

The method of preparing a compound (I) of the present invention will nowbe described.

(1) Synthesis of Common Intermediate (IIB-3)

A compound (IIB-3) can be prepared, for example, through the followingscheme (Scheme 1):

wherein R⁵ represents a hydrogen atom or a tert-butyldimethylsilanyloxygroup.

First, an azide group of a compound (VII) [see Synthesis, No. 9, pp1305-1309 (2000)] is reduced and then the compound is reacted withp-benzyloxybenzenesulfonyl chloride to obtain a compound (VI). Forexample, the azide group is reduced in ethyl acetate, lower alcohol or1,4-dioxane under hydrogen gas flow or pressure at a temperature of roomtemperature to 60° C. in the presence of a catalyst such as 10%palladium-carbon, 20% palladium hydroxide-carbon or platinum. Thesubsequent reaction with p-benzyloxybenzenesulfonyl chloride is usuallyconducted by stirring in an inert solvent such as DMF at a temperatureof 0° C. to room temperature for 1 to 24 hours in the presence of a basesuch as DMAP.

Then, a terminal acetonide group of the compound (VI) is selectivelycleaved and removed. The reaction can be conducted, for example, bystirring in a solvent such as methanol at room temperature to 50° C. for5 hours to 4 days in the presence of a cation exchange resin, optionallyadding water. In this case, the reaction can be also conducted by thefollowing method, in addition to the above-mentioned method using thecation exchange resin. For example, there can be exemplified a method ofstirring in acetonitrile at room temperature for 0.5 to 2 hours, usingcerium chloride heptahydrate and oxalic acid, to obtain the objectiveproduct (V).

Then, only a primary hydroxyl group of the compound (V) is selectivelyconverted into a mesyl group. The reaction is conducted, for example, bystirring in a solvent such as methylene chloride preferably at a lowtemperature of −60 to −20° C. for 30 minutes to 5 hours in the presenceof a base such as triethylamine or DIEA, using 0.95 to 1.05 mols ofmesyl chloride, to obtain the objective compound (IV).

Then, a compound (III) is obtained by the intramolecular ring closurereaction of the compound (IV). The reaction is usually conducted in aninert solvent such as DMF at room temperature to 100° C., preferably 40to 60° C., for 1 to 5 hours in the presence of a base such as potassiumcarbonate or triethylamine to obtain the objective compound (III).

Finally, the compound (III) is debenzylated by hydrogenolysis andreacted with a bromide (VIII) to obtain the objective commonintermediate (IIB-3). This hydrogenolysis is usually conducted bystirring in ethyl acetate, a lower alcohol such as methanol, or1,4-dioxane under hydrogen gas flow or pressure at a temperature of roomtemperature to 60° C. for 1 to 5 hours in the presence of a catalystsuch as 10% palladium-carbon, 20% palladium hydroxide-carbon orplatinum, optionally adding water. The subsequent reaction with thecompound (VIII) is usually conducted in a solvent such as methylenechloride at a temperature of room temperature to 50° C. for 5 to 24hours in the presence of silver oxide to obtain the objective compound(IIB-3).

(2) Preparation of 4,5-dihydroxy Compound (Ia)

A compound (Ia) can be prepared, for example, through the followingscheme (Scheme 2):

wherein R⁶ represents a C₁-C₈ alkyl group, a C₃-C₈ alkenyl group or abenzyl group, Y represents a bromine atom or an iodine atom, and R³ andR⁵ are as defined above.

First, an acetonide group of the common intermediate (IIB-3) shown inScheme 1 is cleaved and removed, and then recombination of the acetonidegroup is conducted to obtain a compound (IIA-3). The cleavage of theacetonide group can be conducted by the above-mentioned method whichuses a cation exchange resin. The reprotection of the acetonide groupcan be conduced, for example, by adding DMP in an inert solvent such asDMF in the presence of an acid catalyst such as p-toluenesulfonic acidmonohydrate or camphor-10-sulfonic acid, and reacting at a temperatureof room temperature to 80° C. for 2 to 24 hours.

Then, the compound (IIA-3) is reacted with a compound (IX) to obtain acompound (IIA-2). The reaction is usually conducted in a solvent such asmethylene chloride at a temperature of room temperature to 50° C. for 24hours to 7 days in the presence of silver oxide to obtain the objectivecompound (IIA-2).

Then, a compound (IIA-1) is obtained by aminolysis of the compound(IIA-2) using hydroxylamine. The reaction is usually conducted bystirring in a lower alcohol such as methanol at room temperature for 24hours to 3 days in the presence of NaCN.

Finally, the acetonide group of the compound (IIA-1) is cleaved andremoved to obtain the objective compound (Ia). The reaction can beconducted by the above-mentioned method which uses a cation exchangeresin. At this time, when R⁵ is a tert-butyldimethylsilanyloxy group,the acetonide group is cleavaged and removed after deprotecting thetert-butyldimethylsilyl group. The deprotection of thetert-butyldimethylsilyl group can be usually conducted by optionallyadding an acid such as acetic acid in an inert solvent such as THF inthe presence of TBAF and reacting at a temperature of room temperatureto 50° C. for 2 to 24 hours.

When R⁶ is a propyl group or a related group thereof, there can be usedanother method shown in the following Scheme 3:

wherein R⁷ represents an allyl group or a substituted allyl group, andR⁸ represents a propyl group or a reduced substituted allyl group.

That is, it is a method of replotecting the acetonide group of thecompound (III), reacting with allyl bromide or substituted allylbromide(XI) and introducing a butynyl group. In the process of cleavage andremoval of a benzyl group in a compound (XII), a carbon-carbon doublebond of the allyl group is reduced, and thus there can be prepared theobjective product (Ia-1) of the formula (Ia) in which R⁶ is a propylgroup or a related group thereof.

(3) Preparation of 3,4-dihydroxy Compound (Ib)

A compound (Ib) can be prepared, for example, through the followingscheme (Scheme 4):

wherein R³, R⁵, R⁶ and Y are as defined above.

First, the common intermediate (IIB-3) shown in Scheme 1 is reacted witha compound (IX) to obtain a compound (IIB-2). The reaction is usuallyconducted in a solvent such as methylene chloride at a temperature ofroom temperature to 50° C. for 24 hours to 7 days in the presence ofsilver oxide to obtain the objective compound (IIB-2).

Then, a compound (IIB-1) is obtained by aminolysis of the compound(IIB-2) using hydroxylamine. The reaction can be conducted by theabove-mentioned method.

Finally, the compound (IIB-1) is deprotected to obtain the objectivecompound (Ib). The deprotection can be conducted in the same manner asshown in Scheme 2.

When R⁶ is a propyl group or a related group thereof in the compound(Ib), another method as shown in Scheme 3 can be used, similar to thecase of the compound (Ia). That is, it is a method of reacting thecompound (III) with allyl bromide or substituted allylbromide (XI) andintroducing a butynyl group. In the process of cleavage and removal of abenzyl group, a carbon-carbon double bond of the allyl group is reduced,and thus there can be prepared the objective product of the formula (Ib)in which R⁶ is a propyl group or a related group thereof.

(4) Preparation of 3,4,5-trihydroxy Compound (Ic)

A compound (Ic) can be prepared, for example, through the followingscheme (Scheme 5):

wherein R³ and R⁵ are as defined above.

First, a compound (IIB-4) is obtained by aminolysis of the commonintermediate (IIB-3) shown in Scheme 1 and hydroxylamine. The reactionis usually conducted by stirring in a lower alcohol such as methanol atroom temperature for 24 hours to 4 days in the presence of NaCN.

Finally, the compound (IIB-4) is deprotected to obtain the objectivecompound (Ic). The reaction can be conducted by the above-mentionedmethod.

These compounds can be administered orally or parenterally to human.

Examples of oral dosage form include solid preparations such as tablets,granules, powders, fine granules and hard capsules; and liquidpreparations such as syrups and soft capsules. These preparations can beprepared by a conventional method. Tablets, granules, powders or finegranules are prepared by mixing the above-mentioned compound orpharmaceutically acceptable salt thereof with commonly usedpharmaceutical additives such as lactose, starch, crystalline cellulose,magnesium stearate, hydroxypropylcellulose and talc, while hard capsulesare prepared by filling these fine granules or powders into a capsule.Syrups are prepared by dissolving or suspending the above-mentionedcompound or pharmaceutically acceptable salt thereof in an aqueoussolution containing sucrose or carboxycellulose, while soft capsules areprepared by dissolving or suspending the above-mentioned compound orpharmaceutically acceptable salt thereof in a lipid excipient such asvegetable oil, oily emulsion or glycol, and filling the resultingsolution or suspension in a soft capsule.

Examples of parenteral dosage form include injections; externalpreparations such as ointments, lotions and creams; suppositorypreparations such as suppositories and pessaries; and pernasalpreparations such as aerosols. These preparations can be prepared by aconventional method and, for example, injections are prepared bydissolving or emulsifying the above-mentioned compound orpharmaceutically acceptable salt thereof in physiological saline or alipid excipient such as vegetable oil, oily emulsion or glycol, andsterilely filling the resulting solution or emulsion in an ampoule or avial. Ointments are prepared, for example, by a conventional method ofadding the above-mentioned compound or pharmaceutically acceptable saltthereof to a base such as vaseline, paraffin or glycerin, and optionallyadding emulsifiers or preservatives.

The dose of the drug of the present invention varies depending on thedosage form, age, sex or weight of patients, or conditions of diseases,but a dairy dose of the active ingredient is usually within a range from0.1 to 600 mg/kg, and preferably from 10 to 200 mg/kg. The drug isadministered once or 2 to 4 times per day.

Test Example 1

Measurement of Matrix Metalloprotease (MMP) Inhibitory Activity

1. Test Compound

Compound (a) of the present invention:(2R,3S,4S,5S)-1-(4′-but-2′-ynyloxybenzenesulfonyl)-4,5-dihydroxy-3-methoxypiperidine-2-carboxylicacid hydroxamide (Compound of Example 2)

Compound (b) of the present invention:(2R,3S,4S,5S)-3-benzyloxy-1-(4′-but-2′-ynyloxybenzenesulfonyl)-4,5-dihydroxypiperidine-2-carboxylicacid hydroxamide (Compound of Example 3)

Compound (c) of the present invention:(2R,3S,4R,5S)-1-(4′-but-2′-ynyloxybenzenesulfonyl)-3,4-dihydroxy-5-methoxypiperidine-2-carboxylicacid hydroxamide (Compound of Example 4)

Compound (d) of the present invention:(2R,3S,4S,5S)-1-(4′-but-2′-ynyloxybenzenesulfonyl)-3,4,5-trihydroxy-piperidine-2-carboxylicacid hydroxamide (Compound of Example 5)

Compound (e) of the present invention:(2R,3S,4S,5S)-1-(4′-but-2′-ynyloxybenzenesulfonyl)-4,5-dihydroxy-3-ethoxypiperidine-2-carboxylicacid hydroxamide (Compound of Example 9)

Compound (f) of the present invention:(2R,3S,4S,5S)-4,5-dihydroxy-1-[4′-(4″-hydroxy-but-2′-ynyloxy)benzenesulfonyl]-3-methoxy-piperidine-2-carboxylicacid hydroxamide (Compound of Example 10)

2. Test Procedure

A DNA fragment coding for catalytic regions in MMP-1 and MMP-9 wasamplified by a PCR method with cDNA prepared from HT-1080 cells derivedfrom human fibrosarcoma. In the same manner, a DNA fragment containing apro-region and a catalyst region in MMP-3 was also prepared. Theamplified DNA fragment was cloned into a commercially availableexpression vector which was designed to allow a histidine tag to add tothe N-terminal, and then transformed into E. coli. The resultingtransformants were cultured and a recombinant MMP was purified from acell lysate using Ni-NTA regin (QIAGEN INC.). Recombinant MMP-3 wasfurther activated by reacting with 1 mM para-aminophenyl mercury acetateat 37° C. for one hour.

The inhibitory activity was measured according to the method by Night etal. [FEBS Lett. 296, p 263 (1992)] or the method by Nagase et al. [J.Biol. Chem. 269, p 20952 (1994)] using a 96-half well black microplate.A solution prepared by dissolving each of test compounds (a) to (f) indimethyl sulfoxide was diluted with a reaction buffer [50 mM Tris-HCl(pH 7.5) buffer containing 150 mM NaCl, 10 mM CaCl₂, 0.05% Brij-35®] andthen 25 μL of the dilute solution was added in a well.

After 25 μL of a MMP solution diluted with the reaction buffer was addedthereto, the mixture was incubated at 37° C. for 10 minutes. Then, thereaction was initiated by adding 50 μL of a 10 μM fluorescence quenchingpeptide substrate prepared from the reaction buffer. As the fluorescencequenching peptide substrate, MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH₂(MCA=7-methoxycoumarin-4-acetyl andDPA=N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl) were used for MMP-1and MMP-9, and MCA-Arg-Pro-Lys-Pro-Val-Nva-Trp-Arg-Lys (DNP)-NH₂(DNP=2,4-dinitrophenyl) was used for MMP-3. A fluorescence intensity(excitation 320/fluoresecence 405 nm) immediately after the initiationof the reaction and a fluorescence intensity after the reaction at 37°C. (for 2 hours for MMP-1 and MMP-3, for 3 hours for MMP-9) weremeasured and a difference in fluorescence intensity was taken as anindicator of enzymatic activity. The inhibition rate (%) at eachconcentration of an inhibitor was calculated and an IC50 value wasdetermined from the dose-response curve. From this IC50 value andpreviously determined Km value for a TACE substrate, a Ki value wascalculated using GraphPad Prism software (GraphPad Software, Inc.).

3. Results

The results are shown in Table 1, along with the results of Test Example2.

Test Example 2

Measurement of Inhibitory Activity Against TNF-α Converting Enzyme(TACE)

1. Test Compounds

the same as in case of Test Example 1

2. Test Procedure

A full length cDNA for TACE was cloned from THP-1 cells derived fromhuman acute monocytic leukemia and a DNA fragment cording a signalregion, a pro-region and a catalytic region was amplified by a PCRmethod. An appropriate restriction enzyme recognizing sequence was addedto both terminals of the amplified fragment and also a sequence of anantigenic octapeptide (FLAG™) tag was inserted in the 3′ terminal. Arecombinant bacmid was prepared from the resulting amplified fragmentusing a pFastBac system (Life Technologies) and then transfected intoinsect cells to generate a recombinant baculovirus. Insect cells wereinfected with the recombinant baculovirus and the recombinant TACEreleased into culture supernatant was purified by an affinity gelcolumn.

The inhibitory activity was measured according to the method by Van Dykeet al. [Bioorg. Med. Chem. Lett. 7, p 1219 (1997)] using a 96 half wellblack microplate. A solution prepared by dissolving each of testcompounds (a) to (f) in dimethylsulfoxide was diluted with a reactionbuffer [20 mM tris-HCl buffer (pH 7.5) containing 0.05% Brij-35®] andthen 40 μL of the dilute solution was added in a well. After 40 μL of aTACE solution (125 ng/mL) diluted with the reaction buffer was addedthereto, the mixture was incubated at 37° C. for 10 minutes. Then, thereaction was initiated by adding 20 μL of a 25 μM fluorescence quenchingpeptide substrateMCA-Pro-Leu-Ala-Glu-Ala-Val-DPA-Arg-Ser-Ser-Ser-Arg-NH₂ prepared fromthe reaction buffer. A fluorescence intensity (excitation 320nm/fluoresecence 405 nm) immediately after the initiation of reactionand a fluorescence intensity after the reaction at 37° C. for 30 minuteswere measured and a difference in fluorescence intensity was taken as anindicator of enzymatic activity. The inhibition rate (%) at eachconcentration of an inhibitor was calculated and an IC50 value wasdetermined from the dose-response curve. From this IC50 value andpreviously determined Km value for a TACE substrate, a Ki value wascalculated using data processing software (GraphPad Prism™, GraphPadSoftware, Inc.).

3. Test Results

The test results are shown in Table 1. TABLE 1 Ki (nM) Test compoundsMMP1 MMP3 MMP9 TACE Compound (a) of the >850 >650 >790 4.3 presentinvention Compound (b) of the >850 >650 >790 9.4 present inventionCompound (c) of the >850 >650 >790 2.4 present invention Compound (d) ofthe >850 490 >790 3.4 present invention Compound (e) ofthe >850 >650 >790 8.5 present invention Compound (f) ofthe >850 >650 >790 18 present invention

Any compound of the present invention selectively exerts an inhibitoryeffect on TACE.

EXAMPLES

The present invention will now be described in detail by way ofReference Examples and Examples.

Reference Example 1 Preparation of4-(bromo-but-2-ynyloxy)-tert-butyldimethylsilane (1)4-(tert-butyl-dimethylsilanyloxy)-but-2-yne-1-ol

But-2-yne-1,4-diol (5 g) was dissolved in DMF (60 mL) and imidazole(11.07 g) was added, followed by the addition of tert-butyldimethylsilylchloride (8.75 g) with stirring under ice cooling and further stirringat room temperature overnight. Ether (500 mL) was added and the reactionsolution was washed with water, and then the organic layer was driedover magnesium sulfate and the solvent was distilled off under reducedpressure. The resulting residue was purified by silica gel mediumpressure column chromatography (ethyl acetate:cyclohexane=1:4→1:3) toobtain the titled compound (4.38 g) as a syrup.

¹H-NMR (CDCl₃) δ: 0.12 (s, 6H), 0.92 (s, 9H), 1.55-1.7 (m, 1H), 4.2-4.3(m, 2H), 4.35 (d, 1H, J=1.0 Hz).

(2) 4-(bromo-but-2-ynyloxy)-tert-butyldimethylsilane

The compound (1.5 g) of (1) was dissolved in methylene chloride (40 mL)and PPh₃ (2.95 g) was added, followed by the addition of carbontetrabromide (3.73 g) and further stirring at room temperature for 30minutes. The solvent was distilled off under reduced pressure and theresulting residue was purified by silica gel medium pressure columnchromatography (ethyl acetate:cyclohexane=1:4) to obtain the titledcompound (2.45 g) as a syrup.

¹H-NMR (CDCl₃) δ: 0.13 (s, 6H), 0.91 (s, 9H), 3.96 (s, 2H), 4.37 (s,2H).

Example 1 Preparation of(3aS,4R,7S,7aS)-5-(4′-but-2′-ynyloxybenzenesulfonyl)-7-hydroxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-4-carboxylicacid methyl ester: (1)(2R,4′R,4″S,5′S)-(benzyloxybenzenesulfonylamino)-(2′,2′,2″,2″-tetramethyl-[4′,4″]bis[[1,3]dioxolanyl]-5′-yl)-aceticacid methyl ester:

A known compound[(2R,4′R,4″S,5′S)-azide-(2′,2′,2″,2″-tetramethyl-[4′,4″]bis[[1,3]dioxolanyl]-5′-yl)-aceticacid methyl ester, 49.5 g] was dissolved in ethyl acetate (300 mL) and10% Pd/C (4.7 g) was added, followed by stirring under hydrogen pressureat 40° C. for 4 hours. The catalyst was removed by filtration and thefiltrate was concentrated under reduced pressure. The resulting residuewas dissolved in DMF (450 mL) and DMAP (19.2 g) andp-benzyloxybenzenesulfonyl chloride (44.4 g) were added, followed bystirring at room temperature overnight. The reaction solution was mixedwith ethyl acetate (700 mL) and then washed in turn with 1N hydrochloricacid, water and saturated saline. The organic layer was dried overmagnesium sulfate and the solvent was distilled off under reducedpressure. The resulting residue was purified by silica gel mediumpressure column chromatography (ethyl acetate:cyclohexane=1:2) to obtainthe titled compound (65 g) as a colorless crystal.

¹H-NMR (CDCl₃) δ: 1.29 (s, 3H), 1.33 (s, 3H), 1.37 (s, 3H), 1.47 (s,3H), 3.52 (s, 3H), 3.9-4.05 (m, 3H), 4.1-4.2 (m, 3H), 5.15 (s, 2H), 5.57(d, 1H, J=6.7 Hz), 7.04 (d, 2H, J=8.9 Hz), 7.35-7.45 (m, 5H), 7.79 (d,2H, J=8.9 Hz).

(2)(1″S,2R,4′S,5′R)-(4-benzyloxybenzenesulfonylamino)-[5′-(1″,2″-dihydroxy-ethyl)-2′,2′-dimethyl-[1,3]dioxolan-4′-yl]-aceticacid methyl ester

The compound (65 g) of (1) was dissolved in acetonitrile (500 mL) andCeCl₃ (90.4 g) and oxalic acid (545 mg) were added, followed by stirringat room temperature for one hour. After adding sodium carbonate untilthe reaction solution is neutralized, the insoluble material was removedby filtration and the residue was washed with ethyl acetate. Thefiltrate and washing were combined and the solvent was distilled offunder reduced pressure. The resulting residue was purified by silica gelmedium pressure column chromatography (ethylacetate:cyclohexane=1:1→3:2, followed by chloroform:methanol=10:1) toobtain the titled compound (29.9 g), and thus a starting material (18.5g) was recovered.

¹H-NMR (CDCl₃)δ: 1.32 (s, 3H), 1.36 (s, 3H), 2.55 (bs, 1H), 3.32 (bs,1H), 3.50 (s, 3H), 3.7-3.8 (m, 2H), 3.85-3.9 (m, 1H), 4.00 (t, 1H, J=7.0Hz), 4.1-4.2 (m, 1H), 4.25-4.3(m, 1H), 5.14(s, 2H), 6.12 (d, 1H, J=7.7Hz), 7.05 (d, 2H, J=8.9 Hz), 7.3-7.5 (m, 5H), 7.78 (d, 2H, J=8.9 Hz).

(3)(1″R,2R,4′S,5′R)-(4-benzyloxybenzenesulfonylamino)-[5′-(1″-hydroxy-2″-methanesulfonyloxy-ethyl)-2′,2′-dimethyl-[1,3]dioxolan-4′-yl]-aceticacid methyl ester

The compound (38.9 g) of (2) was dissolved in methylene chloride (320mL) and triethylamine (8.7 g) was added. The mixture was cooled to −40°C. and mesyl chloride (8.96 g)/methylene chloride (10 mL) were rapidlyadded dropwise, followed by stirring at the same temperature for 30minutes. The reaction solution was washed with saturated saline and theorganic layer was dried over magnesium sulfate, and then the solvent wasdistilled off under reduced pressure. The resulting residue was purifiedby silica gel medium pressure column chromatography (ethylacetate:cyclohexane=1:2→1:1→2:1→4:1) to obtain the titled compound (19.8g), and thus a raw material (9.9 g) was recovered.

¹H-NMR (CDCl₃)δ: 1.31 (s, 3H), 1.36 (s, 3H), 3.12 (s, 3H), 3.54 (s, 3H),3.85-3.95 (m, 1H), 3.97 (dd, 1H, J=6.8, 8.6 Hz), 4.17 (dd, 1H, J=4.8,8.4 Hz), 4.26 (dd, 1H, J=4.9, 6.7 Hz), 4.30 (dd, 1H, J=6.1, 11.1 Hz),4.50 (dd, 1H, J=2.3, 11.1 Hz), 5.15 (s, 2H), 5.73 (d, 1H, J=8.4 Hz),7.06 (d, 2H, J=9.0 Hz), 7.3-7.5 (m, 5H), 7.78 (d, 2H, J=9.0 Hz).

(4)(3aS,4R,7S,7aS)-5-(4′-benzyloxybenzenesulfonyl)-7-hydroxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-4-carboxylicacid methyl ester

The compound (24.1 g) of (3) was dissolved in DMF (200 mL) and potassiumcarbonate (5.8 g) was added, followed by stirring at 50° C. for 1.5hours. The reaction solution was mixed with ethyl acetate (700 mL) andthen washed with water (×2) and saturated saline. The organic layer wasdried over magnesium sulfate and the solvent was distilled off underreduced pressure. The resulting residue was purified by silica gelmedium pressure column chromatography (ethylacetate:cyclohexane=1:2→2:3) to obtain the titled compound (12.8 g) as asyrup.

¹H-NMR (CDCl₃)δ: 1.44 (s, 3H), 1.46 (s, 3H), 2.27 (bs, 1H), 3.21 (dd,1H, J=5.8, 14.7 Hz), 3.48 (dd, 1H, J=4.5, 9.8 Hz), 3.72 (s, 3H), 3.88(d, 1H, J=7.2, 14.7 Hz), 4.05-4.2 (m, 1H), 4.35-4.4 (m, 1H), 4.43 (d,1H, J=9.0 Hz), 5.13 (s, 2H), 7.05 (d, 2H, J=8.9 Hz), 7.3-7.45 (m, 5H),7.79 (d, 2H, J=8.9 Hz).

(5)(3aS,4R,7S,7aS)-5-(4′-but-2′-ynyloxybenzenesulfonyl)-7-hydroxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-4-carboxylicacid methyl ester

The compound (2.13 g) of (4) was dissolved in ethyl acetate (20 mL) and10% Pd/C (200 mg) was added, followed by stirring under hydrogenatmosphere at room temperature for 4 hours. The catalyst was removed byfiltration and the filtrate was concentrated under reduced pressure. Theresulting residue was dissolved in methylene chloride (30 mL) and1-bromo-2-butyne (655 mg) and silver oxide (1.35 g) were added, followedby stirring under nitrogen atmosphere overnight. The insoluble materialwas removed by filtration and the filtrate was concentrated underreduced pressure. The resulting residue was purified by silica gelmedium pressure column chromatography (ethylacetate:cyclohexane=1:2→2:3) to obtain the titled compound (2.3 g) as asyrup.

¹H-NMR (CDCl₃)δ: 1.46 (s, 3H), 1.48 (s, 3H), 1.88 (t, 3H, J=2.3 Hz),2.24 (s, 1H), 3.23 (dd, 1H, J=5.8, 14.7 Hz), 3.50 (dd, 1H, J=4.5, 9.8Hz), 3.76 (s, 3H), 4.05-4.2 (m, 2H), 4.4-4.45 (m, 1H), 4.46 (d, 1H,J=9.0 Hz), 4.73 (q, 1H, J=2.3 Hz), 7.07 (d, 2H, J=9.0 Hz), 7.82 (d, 2H,J=9.0 Hz).

Example 2 Preparation of(2R,3S,4S,5S)-1-(4′-but-2′-ynyloxybenzenesulfonyl)-4,5-dihydroxy-3-methoxypiperidine-2-carboxylicacid hydroxamide (1)(3aS,6R,7S,7aR)-5-(4′-but-2′-ynyloxybenzenesulfonyl)-7-hydroxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-6-carboxylicacid methyl ester

The compound (4.3 g) of Example 1 was dissolved in methanol (40 mL) anda cation exchange resin (MUROMAC, 8.5 g) was added, followed by stirringat room temperature overnight. The insoluble material was removed byfiltration and the filtrate was concentrated under reduced pressure. Theresulting residue was dissolved in DMF (50 mL) and DMP (10 g) andp-toluenesulfonic acid mononhydrate (150 mg) were added, followed bystirring at room temperature overnight and further stirring at 50° C.for 4.5 hours. The reaction solution was distilled off under reducedpressure and the resulting residue was purified by silica gel mediumpressure column chromatography (ethyl acetate:n-hexane=2:3→1:1) toobtain the titled compound (3.48 g) as a syrup.

¹H-NMR (CDCl₃)δ: 1.39 (s, 3H), 1.59 (s, 3H), 1.88 (t, 3H, J=2.3 Hz),2.50 (d, 1H, J=6.5 Hz), 3.23 (dd, 1H, J=9.2, 13.6 Hz), 3.66 (s, 3H),3.87 (dd, 1H, J=7.3, 13.6 Hz), 4.1-4.2 (m, 2H), 4.35-4.45 (m, 1H),4.45-4.55 (m, 1H), 4.73 (q, 2H, J=2.3 Hz), 7.05 (d, 2H, J=8.9 Hz), 7.81(d, 2H, J=8.9 Hz).

(2)(3aS,6R,7S,7aR)-5-(4′-but-2′-ynyloxybenzenesulfonyl)-7-methoxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-6-carboxylicacid methyl ester

The compound (960 mg) of (1) was dissolved in methylene chloride (18 mL)and methyl iodide (3.1 g) and silver oxide (1.51 g) were added, followedby stirring under nitrogen atmosphere at room temperature for 5 days.The insoluble material was removed by filtration and the filtrate wasconcentrated under reduced pressure. The resulting residue was purifiedby silica gel medium pressure column chromatography (ethylacetate:n-hexane=1:3→1:2) to obtain the titled compound (775 mg) as asyrup.

¹H-NMR (CDCl₃)δ: 1.30 (s, 3H), 1.39 (s, 3H), 1.88 (t, 3H, J=2.3 Hz),3.26 (dd, 1H, J=8.0, 13.3 Hz), 3.48 (s, 3H), 3.63 (dd, 1H, J=6.6, 13.3Hz), 3.69 (s, 3H), 4.1-4.2 (m, 2H), 4.25-4.35 (m, 1H), 4.72 (q, 2H,J=2.3 Hz), 4.79 (d, 1H, J=3.0 Hz), 7.05 (d, 2H, J=9.0 Hz), 7.86 (d, 2H,J=9.0 Hz).

(3)(3aS,6R,7S,7aR)-5-(4′-but-2′-ynyloxybenzenesulfonyl)-7-methoxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-6-carboxylicacid hydroxamide

The compound (730 mg) of (2) was dissolved in methanol (12 mL) and anaqueous 50% hydroxylamine solution (3 mL) and sodium cyanide (79 mg)were added, followed by stirring at room temperature for 4 days. Thereaction solution was distilled off under reduced pressure and theresulting residue was purified by silica gel medium pressure columnchromatography (chloroform:methanol=60:1→50:1→40:1) and thenfreeze-dried to obtain the titled compound (356 mg) as an amorphous.

¹H-NMR (DMSO-d₆)δ: 1.18 (s, 3H), 1.36 (s, 3H), 1.84 (s, 3H), 3.25 (s,3H), 3.2-3.35 (m, 1H), 3.6-3.75 (m, 3H), 3.76 (d, 1H, J=7.1 Hz),4.05-4.15 (m, 1H), 4.87 (d, 2H, J=2.2 Hz), 7.12 (d, 2H, J=8.9 Hz), 7.75(d, 2H, J=8.9 Hz), 9.05 (s, 1H), 10.61 (s, 1H).

TOF-Mass: 477 (M+Na), 493 (M+K)

[α]_(D) 36° (c=0.1, MeOH)

(4)(2R,3S,4S,5S)-1-(4′-but-2′-ynyloxybenzenesulfonyl)-4,5-dihydroxy-3-methoxypiperidine-2-carboxylicacid hydroxamide

The compound (270 mg) of (3) was dissolved in methanol (10 mL) and acation exchange resin (MUROMAC, 1.1 g) was added, followed by stirringat room temperature for 2 days. The insoluble material was removed byfiltration and the filtrate was concentrated under reduced pressure. Theresulting residue was purified by silica gel medium pressure columnchromatography (chloroform:methanol=50:1→30:1→10:1) and thenfreeze-dried to obtain the titled compound (111 mg) as an amorphous.

¹H-NMR (DMSO-d₆)δ: 1.84 (t, 3H, J=2.3 Hz), 3.16 (s, 3H), 3.61 (bs, 1H),3.68 (bs, 1H), 4.37 (s, 1H), 4.74 (bs, 1H), 4.84 (q, 2H, J=2.3 Hz), 4.91(bs, 1H), 7.05 (d, 2H, J=8.9 Hz), 7.72 (d, 2H, J=8.9 Hz), 8.87 (bs, 1H),10.63 (s, 1H).

TOF-Mass: 437 (M+Na), 453 (M+K)

[α]_(D) 45° (c=0.1, MeOH)

Example 3 Preparation of(2R,3S,4S,5S)-3-benzyloxy-1-(4′-but-2′-ynyloxybenzenesulfonyl)-4,5-dihydroxypiperidine-2-carboxylicacid hydroxamide (1)(3aS,6R,7S,7aR)-7-benzyloxy-5-(4′-but-2′-ynyloxybenzenesulfonyl)-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-6-carboxylicacid methyl ester

The compound (1.59 g) of Example 2 (1) was dissolved in methylenechloride (15 mL) and benzyl bromide (1.86 g) and silver oxide (3.34 g)were added, followed by stirring under nitrogen atmosphere at roomtemperature for 6 days. The insoluble material was removed by filtrationand the filtrate was concentrated under reduced pressure. The resultingresidue was purified by silica gel medium pressure column chromatography(ethyl acetate:n-hexane=1:3→1:2) to obtain the titled compound (1.75 g)as a syrup.

¹H-NMR (CDCl₃)δ: 1.29 (s, 3H), 1.39 (s, 3H), 1.87 (t, 3H, J=2.3 Hz),3.32 (dd, 1H, J=7.7, 13.3 Hz), 3.63 (dd, 1H, J=6.3, 13.3 Hz), 3.68 (s,3H), 4.19 (dd, 1H, J=3.4, 5.9 Hz), 4.25-4.35 (m, 1H), 4.37 (t, 1H, J=3.3Hz), 4.58 (d, 1H, J=11.4 Hz), 4.68 (q, 2H, J=2.3 Hz), 4.76 (d, 1H,J=11.4 Hz), 4.85 (d, 1H, J=3.2 Hz), 6.97 (d, 2H, J=9.0 Hz), 7.3-7.4 (m,5H), 7.85 (d, 2H, J=9.0 Hz).

(2)(3aS,6R,7S,7aR)-7-benzyloxy-5-(4′-but-2′-ynyloxybenzenesulfonyl)-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-6-carboxylicacid hydroxamide

The compound (1.75 g) of (1) was dissolved in methanol (25 mL) and anaqueous 50% hydroxylamine solution (5 mL) and sodium cyanide (162 mg)were added, followed by stirring at room temperature for 3 days. Thereaction solution was distilled off under reduced pressure and theresulting residue was purified by silica gel medium pressure columnchromatography (chloroform:methanol=30:1) and then freeze-dried toobtain the titled compound (247 mg) as an amorphous, and thus a rawmaterial (252 mg) was recovered.

¹H-NMR (DMSO-d₆)δ: 1.20 (s, 3H), 1.36 (s, 3H), 1.81 (t, 3H, J=2.3 Hz),3.63 (dd, 1H, J=5.7, 13.6 Hz), 3.75-3.85 (m, 1H), 3.9-3.95 (m, 2H),4.15-4.25 (m, 1H), 4.43 (d, 1H, J=11.5 Hz), 4.56 (d, 1H, J=11.5 Hz),4.83 (q, 2H, J=2.3 Hz), 7.11 (d, 2H, J=8.9 Hz), 7.2-7.4 (m, 5H), 7.83(d, 2H, J=8.9 Hz), 9.08 (s, 1H), 10.56 (s, 1H).

TOF-Mass: 553 (M+Na), 569 (M+K)

[α]_(D) 13° (c=0.1, MeOH)

(3)(2R,3S,4S,5S)-3-benzyloxy-1-(4′-but-2′-ynyloxybenzenesulfonyl)-4,5-dihydroxypiperidine-2-carboxylicacid hydroxamide

The compound (180 mg) of (2) was dissolved in methanol (15 mL) and acation exchange resin (MUROMAC, 1 g) was added, followed by stirring atroom temperature overnight and further stirring at 50° C. for 3 hours.The insoluble material was removed by filtration and the filtrate wasconcentrated under reduced pressure. The resulting residue was purifiedby silica gel medium pressure column chromatography(chloroform:methanol=50:1→40:1→30:1→20:1→10:1) and then freeze-dried toobtain the titled compound (59 mg) as an amorphous.

¹H-NMR (DMSO-d₆)δ: 1.82 (s, 3H), 3.67 (s, 1H), 3.92 (s, 1H), 4.48 (d,1H, J=12.0 Hz), 4.56 (d, 1H, J=12.0 Hz), 4.77 (bs, 2H), 6.92 (d, 2H,J=8.9 Hz), 7.2-7.4 (m, 5H), 7.71 (d, 2H, J=8.9 Hz), 8.91 (s, 1H), 10.65(s, 1H).

TOF-Mass: 513 (M+Na), 529 (M+K)

[α]_(D) 11° (c=0.1, MeOH)

Example 4 Preparation of(2R,3S,4R,5S)-1-(4′-but-2′-ynyloxybenzenesulfonyl)-3,4-dihydroxy-5-methoxypiperidine-2-carboxylicacid hydroxamide (1)(3aS,4R,7S,7aS)-5-(4′-but-2′-ynyloxybenzenesulfonyl)-7-methoxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-4-carboxylicacid methyl ester

The compound (1.0 g) of Example 1 was dissolved in methylene chloride(15 mL) and methyl iodide (3.2 g) and silver oxide (1.58 g) were added,followed by stirring under nitrogen atmosphere at room temperature for 4days. The insoluble material was removed by filtration and the filtratewas concentrated under reduced pressure. The resulting residue waspurified by silica gel medium pressure column chromatography (ethylacetate:cyclohexane=1:3→1:2) to obtain the titled compound (852 mg) as asyrup.

¹H-NMR (CDCl₃)δ: 1.44 (s, 3H), 1.47 (s, 3H), 1.88 (t, 3H, J=2.3 Hz),3.38 (dd, 1H, J=5.1, 14.3 Hz), 3.45 (s, 3H), 3.5-3.6 (m, 3H), 3.79 (s,3H), 3.95-4.0 (m, 2H), 4.73 (q, 2H, J=2.3 Hz), 7.08 (d, 2H, J=9.0 Hz),7.83 (d, 2H, J=9.0 Hz).

(2)(3aS,4R,7S,7aS)-5-(4′-but-2′-ynyloxybenzenesulfonyl)-7-methoxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-4-carboxylicacid hydroxamide

The compound (852 mg) of (1) was dissolved in methanol (20 mL) and anaqueous 50% hydroxylamine solution (5 mL) and sodium cyanide (92 mg)were added, followed by stirring at room temperature overnight. Thereaction solution was distilled off under reduced pressure and theresulting residue was purified by silica gel medium pressure columnchromatography (chloroform:methanol=30:1) and then freeze-dried toobtain the titled compound (730 mg) as an amorphous.

¹H-NMR (DMSO-d₆)δ: 1.23 (s, 3H), 1.28 (s, 3H), 1.84 (t, 3H, J=2.2 Hz),2.96 (dd, 1H, J=4.0, 10.0 Hz), 3.25-3.45 (m, 2H), 3.72 (d, 1H, J=8.7Hz), 3.75 (d, 1H, J=6.5 Hz), 4.02 (t, 1H, J=9.5 Hz), 4.88 (t, 2H, J=2.2Hz), 7.17 (d, 2H, J=8.9 Hz), 7.83 (d, 2H, J=8.9 Hz), 9.10 (s, 1H), 10.93(s, 1H).

TOF-Mass: 477 (M+Na), 493 (M+K)

[α]_(D) 33° (c=0.1, MeOH)

(3)(2R,3S,4R,5S)-1-(4′-but-2′-ynyloxybenzenesulfonyl)-3,4-dihydroxy-5-methoxypiperidine-2-carboxylicacid hydroxamide

The compound (500 mg) of (2) was dissolved in methanol (15 mL) and acation exchange resin (MUROMAC, 2 g) was added, followed by stirring atroom temperature overnight. The insoluble material was removed byfiltration and the filtrate was concentrated under reduced pressure. Theresulting residue was purified by silica gel medium pressure columnchromatography (chloroform:methanol=50:1→40:1→30:1→20:1→10:1) and thenfreeze-dried to obtain the titled compound (370 mg) as an amorphous.

¹H-NMR (DMSO-d₆)δ: 1.84(t, 3H, J=2.2 Hz), 3.14 (s, 3H), 3.2-3.5 (m, 4H),3.48 (s, 1H), 3.70 (s, 1H), 3.78 (s, 1H), 4.84 (bs, 2H), 4.91 (bs, 1H),5.29 (bs, 1H), 7.08 (d, 2H, J=8.9 Hz), 7.79 (d, 2H, J=8.9 Hz), 8.83 (s,1H), 10.55 (s, 1H).

TOF-Mass: 437 (M+Na), 453 (M+K)

[α]_(D) −4° (c=0.1, MeOH)

Example 5 Preparation of(2R,3S,4S,5S)-1-(4′-but-2′-ynyloxybenzenesulfonyl)-3,4,5-trihydroxy-piperidine-2-carboxylicacid hydroxamide (1)(3aS,4R,7S,7aS)-5-(4′-but-2′-ynyloxybenzenesulfonyl)-7-hydroxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-4-carboxylicacid hydroxamide

The compound (480 mg) of Example 1 was dissolved in methanol (8 mL) andan aqueous 50% hydroxylamine solution (3 mL) and sodium cyanide (54 mg)were added, followed by stirring at room temperature overnight. Thereaction solution was distilled off under reduced pressure and theresulting residue was purified by silica gel medium pressure columnchromatography (chloroform:methanol=50:1→40:1→30:1) and thenfreeze-dried to obtain the titled compound (210 mg) as an amorphous.

¹H-NMR (DMSO-d₆)δ: 1.22 (s, 3H), 1.28 (s, 3H), 1.83 (t, 3H, J=2.1 Hz),2.76 (dd, 1H, J=4.5, 10.0 Hz), 3.19 (dd, 1H, J=6.0, 14.6 Hz), 3.74 (d,1H, J=8.9 Hz), 3.80 (dd, 1H, J=6.9, 14.5 Hz), 4.0-4.1 (m, 2H), 4.8-4.95(m, 2H), 5.2 (d, 1H, J=3.7 Hz), 7.16 (d, 2H, J=8.9 Hz), 7.84 (d, 2H,J=8.9 Hz), 9.09 (s, 1H), 10.93 (s, 1H).

TOF-Mass: 463 (M+Na), 479 (M+K)

[α]_(D) 52° (c=0.1, MeOH)

(2)(2R,3S,4S,5S)-1-(4′-but-2′-ynyloxybenzenesulfonyl)-3,4,5-trihydroxy-piperidine-2-carboxylicacid hydroxamide

The compound (156 mg) of (1) was dissolved in methanol (8 mL) and acation exchange resin (MUROMAC, 1 g) was added, followed by stirring atroom temperature overnight. The insoluble material was removed byfiltration and the filtrate was concentrated under reduced pressure. Theresulting residue was purified by silica gel medium pressure columnchromatography (chloroform:methanol=30:1→10:1→5:1) and then freeze-driedto obtain the titled compound (120 mg) as an amorphous.

¹H-NMR (DMSO-d₆)δ: 1.84(s, 3H), 3.19 (dd, 1H, J=4.5, 12.8 Hz), 3.4-3.6(m, 2H), 4.05 (s, 1H), 4.26 (s, 1H), 4.7 (bs, 1H), 4.83 (s, 2H), 5.27(s, 1H), 7.07 (d, 2H, J=8.7 Hz), 7.80 (d, 2H, J=8.7 Hz), 8.86 (bs, 1H),10.59 (s, 1H).

TOF-Mass: 423 (M+Na), 439 (M+K)

[α]_(D) 3° (c=0.1, MeOH)

Example 6 Preparation of(3aS,6R,7S,7aR)-5-(4′-but-2′-ynyloxybenzenesulfonyl)-7-hydroxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-6-carboxylicacid hydroxamide

The compound (642 mg) of Example 2 (1) was dissolved in methanol (30 mL)and an aqueous 50% hydroxylamine solution (6 mL) and sodium cyanide (72mg) were added, followed by stirring at room temperature overnight. Thereaction solution was distilled off under reduced pressure and theresulting residue was purified by silica gel medium pressure columnchromatography (chloroform:methanol=50:1→30:1) and then freeze-dried toobtain the titled compound (350 mg) as an amorphous.

¹H-NMR (DMSO-d₆)δ: 1.17 (s, 3H), 1.34 (s, 3H), 1.83 (s, 3H), 3.16 (d,1H, J=8.2 Hz), 3.74 (dd, 1H, J=6.3, 13.8 Hz), 3.83 (dd, 1H, J=7.9, 13.5Hz), 4.0-4.1 (m, 1H), 4.8-4.9 (m, 2H), 5.76 (d, 1H, J=5.5 Hz), 7.14 (d,2H, J=8.9 Hz), 7.83 (d, 2H, J=8.9 Hz), 8.98 (s, 1H), 10.56 (s, 1H).

TOF-Mass: 463 (M+Na), 479 (M+K)

[α]_(D) 50° (c=0.1, MeOH)

Example 7 Preparation of(2R,3S,4S,5S)-3,4,5-trihydroxy-1-[4′-(4″-hydroxy-but-2′-ynyloxy)benzenesulfonyl]-piperidine-2-carboxylicacid hydroxamide (1)(3aS,4R,7S,7aS)-5-[4′-[4″-(tert-butyldimethylsilanyloxy)-but-2′-ynyloxy]benzenesulfonyl]-7-hydroxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-4-carboxylicacid methyl ester

The compound (767 mg) of Example 1 (4) was dissolved in ethyl acetate(20 mL) and 10% Pd/C (92 mg) was added, followed by stirring underhydrogen atmosphere at 40° C. for 2.5 hours. The catalyst was removed byfiltration and the filtrate was concentrated under reduced pressure. Theresulting residue was dissolved in methylene chloride (12 mL) and(4-bromo-but-2-ynyloxy)-tert-butyldimethylsilane (550 mg) and silveroxide (484 mg) were added, followed by stirring under nitrogenatmosphere overnight. Furthermore,(4-bromo-but-2-ynyloxy)-tert-butyldimethylsilane (366 mg) and silveroxide (322 mg) were added, followed by stirring at 50° C. for 3 hours.The insoluble material was removed by filtration and the filtrate wasconcentrated under reduced pressure. The resulting residue was purifiedby silica gel medium pressure column chromatography (ethylacetate:cyclohexane=1:3→2:3) to obtain the titled compound (393 mg) as asyrup.

¹H-NMR (CDCl₃)δ: 0.09 (s, 6H), 0.89 (s, 9H), 1.45 (s, 3H), 1.47 (s, 3H),2.26 (s, 1H), 3.22 (dd, 1H, J=5.7, 14.7 Hz), 3.49 (dd, 1H, J=4.3, 9.7Hz), 3.75 (s, 3H), 3.85 (dd, 1H, J=7.1, 14.7 Hz), 4.05-4.15 (m, 1H),4.35 (s, 2H), 4.37-4.42 (m, 1H), 4.43 (d, 1H, J=9.0 Hz), 4.80 (s, 2H),7.06 (d, 2H, J=8.9 Hz), 7.81 (d, 2H, J=8.9 Hz).

TOF-Mass: 592 (M+Na), 608 (M+K)

(2)(3aS,4R,7S,7aS)-5-[4′-[4″-(tert-butyldimethylsilanyloxy)-but-2′-ynyloxy]benzenesulfonyl]-7-hydroxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-4-carboxylicacid hydroxamide

The compound (378 mg) of (1) was dissolved in methanol (15 mL) and anaqueous 50% hydroxylamine solution (3 mL) and sodium cyanide (32 mg)were added, followed by stirring at room temperature overnight. Thereaction solution was distilled off under reduced pressure and theresulting residue was purified by silica gel medium pressure columnchromatography (chloroform:methanol=50:1→30:1) and then freeze-dried toobtain the titled compound (227 mg) as an amorphous.

¹H-NMR (DMSO-d₆)δ: 0.04 (s, 6H), 0.84 (s, 9H), 1.22 (s, 3H), 1.28 (s,3H), 2.78 (dd, 1H, J=4.4, 9.6 Hz), 3.19 (dd, 1H, J=5.8, 14.5 Hz), 3.34(s, 3H), 3.7-3.8 (m, 2H), 4.0-4.1 (m, 2H), 4.35 (s, 2H), 4.99 (s, 2H),5.23 (d, 1H, J=4.6 Hz), 7.17 (d, 2H, J=8.9 Hz), 7.82 (d, 2H, J=8.9 Hz),9.11 (s, 1H), 10.92 (s, 1H).

TOF-Mass: 593 (M+Na), 609 (M+K)

(3)(3aS,4R,7S,7aS)-7-hydroxy-5-[4′-(4″-hydroxy-but-2′-ynyloxy)benzenesulfonyl]-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-4-carboxylicacid hydroxamide

The compound (217 mg) of (2) was dissolved in THF (5 mL) and acetic acid(62 mg) was added, followed by the addition of TBAF (1 mL) and furtherstirring at room temperature for 3 hours. The reaction solution wasdistilled off under reduced pressure and the resulting residue waspurified by silica gel medium pressure column chromatography(chloroform:methanol=50:1→25:1→10:1) and then freeze-dried to obtain thetitled compound (151 mg) as an amorphous.

¹H-NMR (DMSO-d₆)δ: 1.22 (s, 3H), 1.28 (s, 3H), 2.79 (dd, 1H, J=4.4, 10.0Hz), 3.19 (dd, 1H, J=5.8, 14.5 Hz), 3.7-3.85 (m, 2H), 4.0-4.1 (m, 2H),4.10 (d, 1H, J=5.9 Hz), 4.97 (s, 2H), 5.23 (d, 1H, J=4.5 Hz), 5.27 (t,1H, J=5.9 Hz), 7.17 (d, 2H, J=8.9 Hz), 7.83 (d, 2H, J=8.9 Hz), 9.11 (s,1H), 10.92 (s, 1H).

TOF-Mass: 479 (M+Na), 495 (M+K)

(4)(2R,3S,4S,5S)-3,4,5-trihydroxy-1-[4′-(4″hydroxy-but-2′-ynyloxy)benzenesulfonyl]-piperidine-2-carboxylicacid hydroxamide

The compound (120 mg) of (3) was dissolved in methanol (10 mL) and acation exchange resin (MUROMAC, 2.4 g) was added, followed by stirringat room temperature overnight. The insoluble material was removed byfiltration and the filtrate was concentrated under reduced pressure. Theresulting residue was purified by silica gel medium pressure columnchromatography (chloroform:methanol=20:1→5:1→4:1) and then freeze-driedto obtain the titled compound (26 mg) as an amorphous.

¹H-NMR (DMSO-d₆)δ: 3.19 (dd, 1H, J=4.7, 12.4 Hz), 3.25-3.35 (m, 1H),3.4-3.6 (m, 2H), 4.05 (bs, 1H), 4.12 (d, 1H, J=5.9 Hz), 4.26 (s, 1H),4.67 (d, 1H, J=6.4 Hz), 4.89 (bs, 1H), 4.93 (s, 1H), 5.25-5.35 (m, 2H),7.09 (d, 2H, J=8.8 Hz), 7.79 (d, 2H, J=8.8 Hz), 8.86 (s, 1H), 10.59 (bs,1H).

TOF-Mass: (M+Na), (M+K)

Example 8 Preparation of(2R,3S,4S,5S)-1-(4′-but-2′-ynyloxybenzenesulfonyl)-4,5-dihydroxy-3-isobutoxypiperidine-2-carboxylicacid hydroxamide (1)(3aS,6R,7S,7aR)-5-(4′-benzyloxybenzenesulfonyl)-7-hydroxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-6-carboxylicacid methyl ester

The compound (4.8 g) of Example 1 (4) was dissolved in methanol (70 mL)and a cation exchange resin (MUROMAC, 10.4 g) was added, followed bystirring at room temperature overnight. The insoluble material wasremoved by filtration and the filtrate was concentrated under reducedpressure. The resulting residue was dissolved in DMF (40 mL) and DMP(10.7 mL) and p-toluenesulfonic acid monohydrate (84 mg) were added,followed by stirring at room temperature overnight. The reactionsolution was mixed with ethyl acetate (100 mL) and then washed with anaqueous saturated sodium hydrogencarbonate solution and saturatedsaline. After drying over magnesium sulfate, the solvent was distilledoff under reduced pressure. The resulting residue was purified by silicagel medium pressure column chromatography (ethylacetate:cyclohexane=2:3) to obtain the titled compound (3.62 g) as asyrup.

¹H-NMR (CDCl₃)δ: 1.27 (s, 3H), 1.37 (s, 3H), 2.92 (bs, 1H), 3.21 (dd,1H, J=8.9, 13.5 Hz), 3.62 (s, 3H), 3.80 (dd, 1H, J=7.1, 13.4 Hz),4.05-4.15 (m, 1H), 4.38 (dt, 1H, J=7.0, 7.8 Hz), 4.52 (bs, 1H), 4.58 (d,1H, J=4.1 Hz), 7.04 (d, 2H, J=8.9 Hz), 7.30-7.45 (m, 5H), 7.80 (d, 2H,J=8.9 Hz).

(2)(3aS,6R,7S,7aR)-5-(4′-benzyloxybenzenesulfonyl)-2,2-dimethyl-7-(2″-methylallyloxy)-hexahydro-[1,3]dioxolo[4,5-c]pyridine-6-carboxylicacid methyl ester

The compound (800 mg) of (1) was dissolved in methylene chloride (12 mL)and 3-bromo-2-methylpentene (1.13 g) and silver oxide (970 mg) wereadded, followed by stirring under nitrogen atmosphere at roomtemperature for 4 days. The insoluble material was removed by filtrationand the filtrate was concentrated under reduced pressure. The resultingresidue was purified by silica gel medium pressure column chromatography(ethyl acetate:n-hexane=1:4→1:3) to obtain the titled compound (754 mg)as a syrup.

¹H-NMR (CDCl₃)δ: 1.31 (s, 3H), 1.39 (s, 3H), 1.76 (s, 3H), 3.29 (dd, 1H,J=7.6, 13.6 Hz), 3.61 (dd, 1H, J=6.3, 13.1 Hz), 3.97 (d, 1H, J=12.2 Hz),4.09 (d, 1H, J=12.2 Hz), 4.18 (dd, 1H, J=3.3, 5.9 Hz), 4.25-4.35 (m,2H), 4.76 (d, 1H, J=3.1 Hz), 4.95 (s, 1H), 4.99 (s, 1H), 5.13 (s, 1H),7.04 (d, 2H, J=8.9 Hz), 7.3-7.5 (m, 5H), 7.86 (d, 2H, J=8.9 Hz).

(3)(3aS,6R,7S,7aR)-5-(4′-but-2′-ynyloxybenzenesulfonyl)-7-isobutoxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-6-carboxylicacid methyl ester

The compound (750 mg) of (2) was dissolved in ethyl acetate (15 mL) and10% Pd/C (100 mg) was added, followed by stirring under hydrogenatmosphere at room temperature for 3 hours. The catalyst was removed byfiltration and the filtrate was concentrated under reduced pressure. Theresulting residue was dissolved in methylene chloride (15 mL) and1-bromo-2-butyne (279 mg) and silver oxide (487 mg) were added, followedby stirring under nitrogen atmosphere overnight. The insoluble materialwas removed by filtration and the filtrate was concentrated underreduced pressure. The resulting residue was purified by silica gelmedium pressure column chromatography (ethyl acetate:n-hexane=1:4→1:3)to obtain the titled compound (502 mg) as a syrup.

¹H-NMR (CDCl₃)δ: 0.90 (d, 6H, J=6.7 Hz), 1.31 (s, 3H), 1.40 (s, 3H),1.75-1.85 (m, 1H), 1.88 (t, 3H, J=2.0 Hz), 3.25-3.35 (m, 2H), 3.43 (dd,1H, J=6.6, 8.7 Hz), 3.57 (dd, 1H, J=6.2, 13.2 Hz), 3.70 (s, 3H), 4.17(dd, 1H, J=3.3, 6.0 Hz), 4.22 (t, 1H, J=3.2 Hz), 4.71 (q, 2H, J=2 Hz),4.73 (d, 1H, J=3.1 Hz), 7.04 (d, 2H, J=8.8 Hz), 7.88 (d, 2H, J=8.8 Hz).

(4)(3aS,6R,7S,7aR)-5-(4′-but-2′-ynyloxybenzenesulfonyl)-7-isobutoxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-6-carboxylicacid hydroxamide

The compound (500 mg) of (3) was dissolved in methanol (15 mL) and anaqueous 50% hydroxylamine solution (3 mL) and sodium cyanide (50 mg)were added, followed by stirring at room temperature overnight. Thereaction solution was distilled off under reduced pressure and theresulting residue was purified by silica gel medium pressure columnchromatography (chloroform:methanol=50:1→40:1→30:1) and thenfreeze-dried to obtain the titled compound (317 mg) as an amorphous.

¹H-NMR (DMSO-d₆)δ: 0.73 (d, 6H, J=6.6 Hz), 1.19 (s, 3H), 1.36 (s, 3H),1.45-1.6 (m, 1H), 1.83 (s, 3H), 3.0-3.1 (m, 1H), 3.2-3.35 (m, 2H), 3.60(dd, 1H, J=5.7, 13.7 Hz), 3.65-3.8 (m, 2H), 4.1-4.2 (m, 1H), 4.87 (s,2H), 7.15 (d, 2H, J=8.0 Hz), 7.82 (d, 2H, J=8.0 Hz), 9.03 (s, 1H), 10.50(s, 1H).

(5)(2R,3S,4S,5S)-1-(4′-but-2′-ynyloxybenzenesulfonyl)-4,5-dihydroxy-3-isobutoxypiperidine-2-carboxylicacid hydroxamide

The compound (275 mg) of (4) was dissolved in methanol (10 mL) and acation exchange resin (MUROMAC, 2 g) was added, followed by stirring atroom temperature for 2 days. The insoluble material was removed byfiltration and the filtrate was concentrated under reduced pressure. Theresulting residue was purified by silica gel medium pressure columnchromatography (chloroform:methanol=50:1→40:1→30:1→10:1) and thenfreeze-dried to obtain the titled compound (49 mg) as an amorphous, andthus a raw material (157 mg) was recovered.

¹H-NMR (DMSO-d₆)δ: 0.75-0.85 (m, 6H), 1.6-1.7 (m, 1H), 1.84 (t, 1H,J=2.3 Hz), 3.12 (dd, 1H, J=7.0, 8.9 Hz), 3.24 (dd, 1H, J=6.0, 9.1 Hz),3.63 (bs, 1H), 3.75 (bs, 1H), 4.34 (bs, 1H), 4.79 (bs, 1H), 4.83 (q, 2H,J=2.3 Hz), 4.92 (bs, 1H), 7.08 (d, 2H, J=8.8 Hz), 7.74 (d, 2H, J=8.8Hz), 8.92 (s, 1H), 10.63 (s, 1H).

Example 9 Preparation of(2R,3S,4S,5S)-1-(4′-but-2′-ynyloxybenzenesulfonyl)-4,5-dihydroxy-3-ethoxypiperidine-2-carboxylicacid hydroxamide (1)(3aS,6R,7S,7aR)-5-(4′-but-2′-ynyloxybenzenesulfonyl)-7-ethoxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-6-carboxylicacid methyl ester

The compound (1.47 g) of Example 2 (1) was dissolved in methylenechloride (15 mL) and ethyl iodide (5.2 g) and silver oxide (2.3 g) wereadded, followed by stirring under nitrogen atmosphere at roomtemperature for 6 days. The insoluble material was removed by filtrationand the filtrate was concentrated under reduced pressure. The resultingresidue was purified by silica gel medium pressure column chromatography(ethyl acetate:n-hexane=1:4→1:3) to obtain the titled compound (485 mg)as a syrup.

¹H-NMR (CDCl₃)δ: 1.19 (t, 3H, J=7.0 Hz), 1.30 (s, 3H), 1.39 (s, 3H),1.88 (t, 3H, J=2.2 Hz), 3.30 (dd, 1H, J=7.6, 13.3 Hz), 3.55-3.65 (m,2H), 3.70 (s, 3H), 3.7-3.8 (m, 1H), 4.15-4.2 (m, 1H), 4.24 (t, 1H, J=3.3Hz), 4.29 (q, 1H, J=6.2 Hz), 4.72 (q, 2H, J=2.2 Hz), 4.73 (d, 1H, J=3.2Hz), 7.04 (d, 2H, J=8.9 Hz), 7.86 (d, 2H, J=8.9 Hz).

(2)(3aS,6R,7S,7aR)-5-(4′-but-2′-ynyloxybenzenesulfonyl)-7-ethoxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-6-carboxylicacid hydroxamide

The compound (480 mg) of (1) was dissolved in methanol (15 mL) and anaqueous 50% hydroxylamine solution (3 mL) and sodium cyanide (50 mg)were added, followed by stirring at room temperature for 2 days. Thereaction solution was distilled off under reduced pressure and theresulting residue was purified by silica gel medium pressure columnchromatography (chloroform:methanol=60:1→40:1→30:1) and thenfreeze-dried to obtain the titled compound (205 mg) as an amorphous.

¹H-NMR (DMSO-d₆)δ: 0.93 (t, 3H, J=7.0 Hz), 1.18 (s, 3H), 1.36 (s, 3H),1.83 (t, 3H, J=1.7 Hz), 3.29 (dd, 1H, J=8.3, 13.4 Hz), 3.5-3.55 (m, 1H),3.6-3.75 (m, 4H), 4.12 (q, 1H, J=7.7 Hz), 4.87 (q, 2H, J=2.2 Hz), 7.15(d, 2H, J=8.9 Hz), 7.82 (d, 2H, J=8.9 Hz), 9.04 (s, 1H), 10.54 (s, 1H).

TOF-Mass: 491 (M+Na), 500 (M+K)

(3)(2R,3S,4S,5S)-1-(4′-but-2′-ynyloxybenzenesulfonyl)-4,5-dihydroxy-3-ethoxypiperidine-2-carboxylicacid hydroxamide

The compound (165 mg) of (2) was dissolved in methanol (15 mL) and acation exchange resin (MUROMAC, 2.5 g) was added, followed by stirringat room temperature for 2 days. The insoluble material was removed byfiltration and the filtrate was concentrated under reduced pressure. Theresulting residue was purified by silica gel medium pressure columnchromatography (chloroform:methanol=50:1→30:1→10:1) and thenfreeze-dried to obtain the titled compound (44 mg) as an amorphous.

¹H-NMR (DMSO-d₆)δ: 1.01 (t, 3H, J=6.0 Hz), 1.84 (t, 3H, J=2.3 Hz),3.45-3.55 (m, 1H), 3.59 (bs, 1H), 3.78 (bs, 1H), 4.35 (bs, 1H), 4.75(bs, 1H), 4.84 (q, 2H, J=2.3 Hz), 4.91 (bs, 1H), 7.07 (d, 2H, J=8.7 Hz),7.74 (d, 2H, J=8.7 Hz), 8.88 (s, 1H), 10.62 (s, 1H).

TOF-Mass: 451 (M+Na), 467 (M+K)

Example 10 Preparation of(2R,3S,4S,5S)-4,5-dihydroxy-1-[4′-(4″-hydroxy-but-2′-ynyloxy)benzenesulfonyl]-3-methoxy-piperidine-2-carboxylicacid hydroxamide (1)(3aS,6R,7S,7aR)-5-(4′-benzyloxybenzenesulfonyl)-2,2-dimethyl-7-methoxy-hexahydro-[1,3]dioxolo[4,5-c]pyridine-6-carboxylicacid methyl ester

The compound (1.03 g) of Example 8 (1) was dissolved in methylenechloride (10 mL) and methyl iodide (1.35 mL) and silver oxide (1.5 g)were added, followed by stirring under nitrogen atmosphere at roomtemperature for 5 days. The insoluble material was removed by filtrationand the filtrate was concentrated under reduced pressure. The resultingresidue was purified by silica gel medium pressure column chromatography(ethyl acetate:cyclohexane=1:4→1:3) to obtain the titled compound (765mg) as a syrup.

¹H-NMR (CDCl₃)δ: 1.29 (s, 3H), 1.37 (s, 3H), 3.24 (dd, 1H, J=7.9, 13.2Hz), 3.46 (s, 3H), 3.61 (dd, 1H, J=6.6, 13.4 Hz), 3.66 (s, 3H), 4.1-4.15(m, 2H), 4.27 (q, 1H, J=6.9 Hz), 4.78 (bs, 1H), 5.12 (s, 2H), 7.04 (d,2H, J=8.5 Hz), 7.3-7.45 (m, 5H), 7.83 (d, 2H, J=8.5 Hz).

(2)(3aS,4R,7S,7aS)-5-[4′-[4″-(tert-butyldimethylsilanyloxy)-but-2′-ynyloxy]benzenesulfonyl]-7-methoxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-6-carboxylicacid methyl ester

The compound (870 mg) of (1) was dissolved in ethyl acetate (25 mL) and10% Pd/C (110 mg) was added, followed by stirring under hydrogenatmosphere at room temperature for 3.5 hours. The catalyst was removedby filtration and the filtrate was concentrated under reduced pressure.The resulting residue was dissolved in methylene chloride (8 mL) and(4-bromo-but-2-ynyloxy)-tert-butyldimethylsilane (700 mg) and silveroxide (616 mg) were added, followed by stirring under nitrogenatmosphere at 50° C. overnight. The insoluble material was removed byfiltration and the filtrate was concentrated under reduced pressure. Theresulting residue was purified by silica gel medium pressure columnchromatography (ethyl acetate:cyclohexane=1:3) to obtain the titledcompound (490 mg) as a syrup.

¹H-NMR (CDCl₃)δ: 0.093 (s, 6H), 0.89 (s, 9H), 1.29 (s, 3H), 1.37 (s,3H), 3.24 (dd, 1H, J=7.9, 13.2 Hz), 3.47 (s, 3H), 3.60 (dd, 1H, J=6.5,13.2 Hz), 4.1-4.2 (m, 1H), 4.27 (q, 1H, J=6.8 Hz), 4.35 (s, 2H), 4.78(bs, 3H), 7.03 (d, 2H, J=8.5 Hz), 7.84 (d, 2H, J=8.5 Hz).

(3)(3aS,4R,7S,7aS)-5-[4′-[4″-(tert-butyldimethylsilanyloxy)-but-2′-ynyloxy]benzenesulfonyl]-7-methoxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-6-carboxylicacid hydroxamide

The compound (487 mg) of (2) was dissolved in methanol (20 mL) and anaqueous 50% hydroxylamine solution (4 mL) and sodium cyanide (41 mg)were added, followed by stirring at room temperature for 2 days. Thereaction solution was distilled off under reduced pressure and theresulting residue was purified by silica gel medium pressure columnchromatography (chloroform:methanol=50:1→35:1) and then freeze-dried toobtain the titled compound (122 mg) as an amorphous.

¹H-NMR (DMSO-d₆)δ: 0.04 (s, 6H), 0.84 (s, 9H), 1.18 (s, 3H), 1.36 (s,3H), 3.25 (s, 3H), 3.2-3.3 (m, 1H), 3.55-3.7 (m, 3H), 3.76 (d, 1H, J=7.2Hz), 4.09 (q, 1H, J=7.5 Hz), 4.35 (s, 2H), 4.99 (s, 2H), 7.16 (d, 2H,J=8.7 Hz), 7.82 (d, 2H, J=8.7 Hz), 9.05 (s, 1H), 10.62 (s, 1H).

TOF-Mass: 607 (M+Na), 623 (M+K)

(4)(3aS,4R,7S,7aS)-5-[4′-(4″-hydroxy-but-2′-ynyloxy)benzenesulfonyl]-7-methoxy-2,2-dimethyl-hexahydro-[1,3]dioxolo[4,5-c]pyridine-6-carboxylicacid hydroxamide

The compound (121 mg) of (3) was dissolved in THF (5 mL) and acetic acid(34 mg) was added, followed by the addition of TBAF (0.57 mL) andfurther stirring at room temperature for 3 hours. The reaction solutionwas distilled off under reduced pressure and the resulting residue waspurified by silica gel medium pressure column chromatography(chloroform:methanol=50:1→25:1→15:1) and then freeze-dried to obtain thetitled compound (70 mg) as an amorphous.

¹H-NMR (DMSO-d₆)δ: 1.19 (s, 3H), 1.36 (s, 3H), 3.25 (s, 3H), 3.2-3.3 (m,1H), 3.62 (t, 1H, J=7.1 Hz), 3.65-3.7 (m, 2H), 3.76 (d, 1H, J=7.2 Hz),4.05-4.15 (m, 3H), 4.97 (s, 2H), 5.26 (t, 1H, J=5.7 Hz), 7.16 (d, 2H,J=8.9 Hz), 7.82 (d, 2H, J=8.9 Hz), 9.05 (s, 1H), 10.61 (s, 1H).

TOF-Mass: 493 (M+Na), 509 (M+K)

(5)(2R,3S,4S,5S)-4,5-dihydroxy-1-[4′-(4″hydroxy-but-2′-ynyloxy)benzenesulfonyl]-3-methoxy-piperidine-2-carboxylicacid hydroxamide

The compound (65 mg) of (4) was dissolved in methanol (6 mL) and acation exchange resin (MUROMAC, 1.5 g) was added, followed by stirringat room temperature overnight. The insoluble material was removed byfiltration and the filtrate was concentrated under reduced pressure. Theresulting residue was purified by silica gel medium pressure columnchromatography (chloroform:methanol=20:1→6:1→5:1) and then freeze-driedto obtain the titled compound (17 mg) as an amorphous.

¹H-NMR (DMSO-d₆)δ: 3.21 (s, 3H), 3.2-3.3 (m, 1H), 3.43 (bs, 1H), 3.61(bs, 1H), 4.11 (d, 1H, J=5.9 Hz), 4.27 (s, 1H), 4.75 (d, 1H, J=4.8 Hz),4.94 (s, 2H), 5.27 (t, 1H, J=6.0 Hz), 7.10 (d, 2H, J=8.7 Hz), 7.73 (d,2H, J=8.7 Hz), 8.86 (s, 1H), 10.63 (bs, 1H).

TOF-Mass: 453 (M+Na), 469 (M+K)

Example 11

Preparation of Tablets

In the following manner, tablets, each of which contains 100 mg of(2R,3S,4R,5S)-1-(4′-but-2′-ynyloxybenzenesulfonyl)-3,4-dihydroxy-5-methoxypiperidine-2-carboxylicacid hydroxamide (compound c) of Example 4, are obtained.

[Formulation] Ingredients Amount Basis (compound c) 100 Parts by weight Cornstarch 46 Parts by weight Microcrystalline cellulose 98 Parts byweight Hydroxypropylcellulose  2 Parts by weight Magnesium stearate  4Parts by weight[Operation]

A basis, cornstarch and microcrystalline cellulose are mixed and asolution prepared by dissolving hydroxypropylcellulose in 50 parts byweight of water is added, followed by well kneading. The resultingkneaded mixture is passed through a sieve, granulated and then dried toobtain granules. The resulting granules are mixed with magnesiumstearate and then the mixture is compressed into tablets each having aweight of 250 mg.

Example 12

Preparation of Granules

In the following manner, granules containing(2R,3S,4R,5S)-1(4′-but-2′-ynyloxybenzenesulfonyl)-3,4-dihydroxy-5-methoxypiperidine-2-carboxylicacid hydroxamide (compound c) of Example 4 are obtained.

[Formulation] Ingredients Amount Basis (compound c) 200 Parts by weightLactose 185 Parts by weight Cornstarch 109 Parts by weightHydroxypropylcellulose  6 Parts by weight[Operation]

A basis, lactose and cornstarch are mixed and a solution prepared bydissolving hydroxypropylcellulose in 120 parts by weight of water isadded, followed by well kneading. The resulting kneaded mixture ispassed through a #20 mesh sieve, granulated, dried and then sized toobtain granules containing 200 mg of a basis in 500 mg.

Example 13

Preparation of Capsules

In the following manner, capsules, each of which contains 100 mg of(2R,3S,4R,5S)-1-(4′-but-2′-ynyloxybenzenesulfonyl)-3,4-dihydroxy-5-methoxypiperidine-2-carboxylicacid hydroxamide (compound c) of Example 4, are obtained.

[Formulation] Ingredients Amount Basis (compound c) 100 Parts by weight Lactose 35 Parts by weight Cornstarch 60 Parts by weight Magnesiumstearate  5 Parts by weight[Operation]

The above respective ingredients are well mixed and each 200 mg of theresulting powder mixture is filled in a capsule to obtain a capsule.

Example 14

Preparation of Injections

A mixture of 0.5 parts by weight of(2R,3S,4R,5S)-1-(4′-but-2′-ynyloxybenzenesulfonyl)-3,4-dihydroxy-5-methoxypiperidine-2-carboxylicacid hydroxamide (compound c) of Example 4 and 5 parts by weight ofsorbitol is dissolved in distilled water for injection to make 100 partsby weight and then the aqueous solution is filtered through a membranefilter. Each 5 g of the filtrate is filled in an ampoule subjected to atreatment of replacing by nitrogen. After melt sealing, the ampoule issterilized at 120° C. for 15 minutes to obtain an injection containing25 mg of a compound c in an ampoule.

Example 15

Preparation of Ointments

1.0 Parts by weight of(2R,3S,4R,5S)-1-(4′-but-2′-ynyloxybenzenesulfonyl)-3,4-dihydroxy-5-methoxypiperidine-2-carboxylicacid hydroxamide (compound c) of Example 4 and 0.1 parts by weight ofbutylparaben are dispersed in 5.0 parts by weight of light liquidparaffin, ground in a mortar and then passed through a #200 mesh sieve.The resulting product is mixed with 5.0 parts by weight of liquidparaffin and then uniformly dispersed in 88.9 parts by weight of gelledhydrocarbon heated to about 60° C. to obtain oily ointments.

INDUSTRIAL APPLICABILITY

The compound of the present invention exerts a potent inhibitory effecton TACE, while it hardly exerts a inhibitory effect on MMP1, MMP3 andMMP9 (see Test Examples). Therefore, the compound of the presentinvention is extremely useful as a TACE inhibitor.

1. A compound represented by the general formula (I):

wherein R¹ and R² represent a hydrogen atom, a C₁-C₈ alkyl group, aC₃-C₈ alkenyl group or a benzyl group and at least one of R¹ and R² is ahydrogen atom, and R³ represents a hydrogen atom or a hydroxyl group, ora pharmaceutically acceptable salt thereof:
 2. A drug comprising thecompound (I) according to claim 1, or a pharmaceutically acceptable saltthereof, as an active ingredient.
 3. A TACE inhibitor comprising thecompound (I) according to claim 1, or a pharmaceutically acceptable saltthereof, as an active ingredient.
 4. A compound represented by thefollowing formula (IIA) or (IIB):

wherein R¹′ and R²′ represent a hydrogen atom, a C₁-C₈ alkyl group, aC₃-C₈ alkenyl group or a benzyl group, R³ represents a hydrogen atom ora hydroxyl group, and CO—R⁴ represents a hydroxamic acid equivalent.